The present invention is directed to a flow cytometric analysis of allergen induced basophil activation. The particular combinations of antibodies labelled with different fluorophores in combination with a new approach to obtain the mean or median fluorescence intensities (MFI) resulting in the provision of an Activation Index which allows for a better correlation of the individual patient results to their clinical history and, therefore, provides a markedly improved clinical sensitivity over existing methods by maintaining a high specificity. Thereby, a novel in vitro allergy test is provided.
Human basophils are one class of leukocytes circulating in the blood stream and belong to the granulocytes. Despite their low abundance in human blood (less than 2% of the leukocyte fraction), they play a central role in allergic hypersensitivity reactions by releasing potent inflammatory mediators. Moreover, basophils represent the major interleukin-4 secreting human cell. Interleukin-4 plays a key immunological role.
Immunoglobulin E (IgE) represents one of the classes of immunoglobulins. It is known to participate in allergic reactions. Circulating IgE molecules bind to the basophil membrane via the high affinity FcεRI receptor. An allergen, usually a protein of a molecular size of more than 5000 Da, is able to crosslink two neighboured IgE molecules bound on the basophils. By this crosslinking, a complex process is activated at the membrane level by an increase of membrane fluidity leading to IgE/FcεRI receptor clustering, degranulation of the cells and initiation of ionic fluxes into the basophilic cells which ultimately lead to the release of inflammatory mediators, such as histamine or sulfidoleukotrienes, as well as to the expression on, up-regulation at or migration of glycoproteins to the basophil membrane. Examples of such glycoproteins are CD45, CD63 or CD203c which are members of the so-called clustered differentiation (CD) antigens.
Apart from the IgE mediated allergic reactions which can be induced by protein allergens, bivalent molecules, monovalent allergens (true haptens) presented to basophils by a carrier macromolecule, anti-IgE antibodies, anti-FcεRI receptor antibodies, etc, so-called non-IgE mediated reactions do also exist or are related to allergens for which an IgE mechanism has not been clearly established. The non-IgE mediated reactions are usually induced by low molecular weight substances such as a whole series of drugs, some food additives, other chemicals or agents, fMLP, complement factor C5a, etc. Both IgE and non-IgE mediated reactions may lead to basophil activation. However, the rate of positive reactions is much lower in non-IgE mediated processes. Oppositely to IgE mediated basophil activation, the underlying pathophysiological mechanism for non-IgE mediated basophil activation and allergic reactions, respectively, is still not known to date.
Basophil activation was first studied by quantification of the amount of mediator release (histamine and leukotriene C4) or by staining with specific fluorescent dyes and subsequent microscopic counting of fluorescent basophils. Later, the availability of flow cytometers capable of analysing several thousand cells per second led to the development of several methods first based on basophil staining with alcian blue (T. Nakagawa, B. M. Stadler, A. L. de Week: Flow-cytometric analysis of human basophil degranulation. I. Quantification of human basophils and their degranulation by flow-cytometry. Allergy 1981, 36, 39-47; T. Nakagawa, O. Moyseyenko, A. L. de Weck: Flow-cytometric analysis of human basophil degranulation. Ill. Degranulation induced by allergens and antibodies in hay fever and bee venom allergic patients. Int Arch Allergy Appl Immunol 1981, 64, 201-209) and later with the use of fluorescently labelled antibodies specifically reacting with various basophilic membrane markers (reviewed by F. Hennersdorf, S. Florian, A. Jakob, K. Baumgartner, K. Sonneck, A. Nordheim, T. Biedermann, P. Valent, H. J. Bühring: Identification of CD13, CD107a, and CD164 as novel basophil activation markers and dissection of two response patterns in time kinetics of IgE-dependent upregulation. Cell Res 2005, 15, 325-335).
Early studies have demonstrated that quantification of allergen induced basophil activation by flow cytometric measurements is possible (P. Gane, C. Pecquet, H. Crespeau, P. Lambin, F. Leynadier, P. Rouger: Flow cytometric monitoring of allergen induced basophil activation. Cytometry 1995, 19, 361-365). The measurements have been carried out with aero-allergens such as protein extracts from grass or tree pollens, cat dander and dust mites. Upon basophil activation the following changes were detected: (i) a decrease in the number of detectable basophils, (ii) a decrease in IgE mean fluorescence intensity (MFI), and (iii) an increase in CD45 MFI. The increase of the CD45 MFI was detected for all aero-allergens tested clearly indicating that expression of the CD45 molecule on basophils is increased. It was concluded that the increase in CD45 expression is a measure of basophil activation and can, therefore, facilitate investigations on allergen induced basophil activation.
Since it turned out that CD45 was not an optimal expression marker to follow basophil activation, the discovery of the basophil activation marker, CD63 (E. F. Knol, F. P. Mul, H. Jansen, J. Calafat, D. Roos: Monitoring human basophil activation via CD63 monoclonal antibody 435. J Allergy Clin Immunol 1991, 88, 328-338), and its first use for analysing allergen induced basophil activation as described by Sainte-Laudy et al. (J. Sainte-Laudy, C. Vallon, J.-C. Guérin: Analyse de l'expression membranaire du marqueur CD63 par activation du basophil humain. Applications au diagnostic allergologique. Allergie et Immunologie 1994, 26, 211-214; A. Funes, J. Sainte-Laudy, A. Sabbah: Methode pour l'analyse de l'activation des basophiles humain par mésure de l'expression membranaire du marqueur CD63. FR 2 765 341-A1) was considered a major breakthrough. Among many others, a study of flow cytometric analysis of insect venom allergy looking at CD63 expression on basophils has demonstrated a perfect correlation with the clinical history of the patients as well as a high correlation to leukotriene release which is a known indicator of basophil activation (J. Sainte-Laudy, A. Sabbah, M. Drouet, M. G. Lauret, M. Loiry: Diagnosis of venom allergy by flow cytometry. Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release. Clin Exp Allergy 2000, 30, 1166-1171). The sensitivity and specificity (both 100%) of the flow cytometry method was better than that of skin tests and specific IgE determination. Hence, the use of flow cytometry based on percentage CD63 expression for the diagnosis of insect venom allergy has been strongly advocated and ever since, this method has been applied to a wide panel of protein and drug allergens (reviewed, by M. L. Sanz, J. P. Maselli, P. M. Gamboa, A. Oehling, I. Diéguez, A. L. de Weck: Flow cytometric basophil activation test: a review. J Investig Allergol Clin Immunol 2002, 12, 143-154; D. G. Ebo, J. Sainte-Laudy, C. H. Bridts, C. H. Mertens, M. M. Hagedorens, A. J. Schuerwegh, L. S. de Clerck, W. J. Stevens: Flow-assisted allergy diagnosis: current applications and future perspectives. Allergy 2006, 61, 1028-1039).
Later, protocols using the increase of CD203c expression as a basophil activation marker for allergy diagnosis have also been described as alternatives to the use of CD63 (eg. I. J. Platz, M. Binder, A. Marxer, G. Lischka, P. Valent, H. J. Bühring: Hymenoptera-venom-induced upregulation of the basophil activation marker ecto-nucleotide pyrophosphatase/phosphodieasterase 3 (CD203c) in sensitized individuals. Int Arch Allergy Immunol 2002, 126, 335-342; R. Boumiza, G. Monneret, M. F. Forissier, J. Savoye, M. C. Gutowski, W. S. Powell, J. Bienvenu: Marked improvement of the basophil activation test by detecting CD203c instead of CD63. Clin Exp Allergy 33, 259-265; A. Ocmant, Y. Peignois, S. Mulier, L. Hanssens, A. Michils, L. Schandene: Flow cytometry for basophil activation markers: the measurement of CD203c upregulation is as reliable as CD63 expression in the diagnosis of cat allergy. J Immunol Methods 2007, 320, 40-48).
Monneret et al (Monneret G et al: Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relexant drugs. Clin Immunol, 2002, 102 (2), 192-199) describe the monitoring of basophil activation using CD63 and/or CCR 3. Data were obtained from flow cytometry which were compared to skin tests, specific IgE and histamine release results. The flow cytometric protocol described by Monneret et al appears to be useful in allergy diagnosis, since it is specific and complementary to specific IgE detection.
Boumiza et al. (Boumiza R, Debard A-L, Monneret G: The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives. Clin Mol Allergy 2005, 3 (9), 1-8) disclose in a review article the recent developments in clinical studies, standardization and perspectives of the flow cytometric technique. The review is focused on flow cytometry as a tool for monitoring basophil activation upon allergen challenge by detecting surface expression of degranulation/activation markers like CD63 or CD203c. Boumiza et al. focus on the use of anti-CD45 antibodies in the presence of CRTH2/DP2 or IgE for basophil recognition together with a specific staining protocol so that the basophil activation test is useful as a tool for in vitro diagnosis of immediate allergy.
Summing up, it has been demonstrated that flow cytometric analysis of basophil activation provides both high sensitivities and high specificities when used with proteins or protein extracts (inhalative allergens, food allergens, insect venoms, latex, and others).
By contrast, results obtained with drugs showed similar specificities but much lower sensitivities below 50% for beta-lactams antibiotics (eg. M. J. Torres, A. Padial, C. Mayorga, T. Fernandez, E. Sanchez-Sabate, J. A. Cornejo-Garcia, C. Antunez, M. Blanca: The diagnostic interpretation of basophil activation test in immediate allergic reactions to betalactams. Clin Exp Allergy 2004, 34, 1768-1775), 12 to 55% for non-steroidal anti-inflammatory drugs (P. Gamboa, M. L. Sanz, M. R. Cabellero, I. Urrutia, I. Antepara, R. Esparza, A. L. de Weck: The flow-cytometric determination of basophil activation induced by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is useful for in vitro diagnosis of the NSAID hypersensitivity syndrome. Clin Exp Allergy 2004, 34 1448-1457) or below 54% for muscle relaxants (eg. N. Abuaf, B. Rajoely, H Ghazouani, D. Levy, C. Pecquet, H Chabane, F. Leynadier: Validation of a flow cytometric assay detecting in vitro basophil activation for the diagnosis of muscle relaxant allergy. J. Allergy Clin Immunol 1999, 104, 411-418), except in patients with perioperative anaphylaxis to muscle relaxants (P. S. Sudheer, J. E. Hall, G. F. Read, A. W. Rowbottom, P. E. Williams: Flowcytometric investigation of peri-anaesthetic anaphylaxis using CD63 and CD203c. Anaesthesia 2005, 60, 251-256). This, however, is not suitable for routine allergy diagnosis.
WO 2007/093517 A1 describes a method and a kit for determining the appearance of adverse reactions in patients in need to undergo to an administration of a pharmaceutical compound. The method for determining potential hypersensitivity in a patient to pseudo-allergic reactions comprises adding a predetermined amount of a compound of anaphylatoxic activity to a sample of the patient's blood and determining the amount of activation of the patient's basophil cells in said blood sample. The compound with anaphylatoxic activity is preferably selected from C3a, C5a, analogs of C3a or C5a, derivatives of C3a or C5a and mixtures thereof. However, this is a very unspecific test, as it does not address the nature of the drug substance potentially causing the anaphylatoxic reaction.
Thus, there is a need for a more sensitive method for allergy diagnosis, in particular for diagnosis of allergies or hypersensitivity to low molecular weight substances such as drugs. J. Sainte-Laudy et al. (Diagnosis of venom allergy by flow cytometry. Correlation with clinical history, skin tests, specific IgE, histamine and leukotriene C4 release. Clin Exp Allergy 2000, 30, 1166-1171) and P. Gane, et al. (Flow cytometric monitoring of allergen induced basophil activation. Cytometry 1995, 19, 361-365) reported that the activation of human basophils led also to a decrease of IgE density on basophil membranes which could be observed by a decrease of the mean fluorescence intensity of the IgE positive cell population (MFI-IgE). In these two documents, the MFI-IgE decrease was observed upon activation with highly allergenic protein extracts from aero-allergens and insect venoms and was just considered as a pharmacological effect on basophilic cells without a use for clinical allergy diagnosis. Based on these observations, two explorative studies have recently been performed to address this phenomenon of IgE down-regulation to drug allergens (J. Sainte-Laudy, A. Boumediene, F. Touraine, I. Orsel, M. Cogné: Analysis of IgE down regulation induced by basophil activation, Application to the diagnosis of muscle relaxant allergic hypersensitivity by flow cytometry. Inflamm Res 2006, 55, Supplement I, S21-S22; J. Sainte-Laudy, F. Touraine, A. Boumediene, F. Bonnaud, M. Cogné: Clinico-biological characteristics of flow cytometry applied to hypersensitivity to NSAIDs. Inflamm Res 2007, 56, Supplement I, S63-S64). It could be shown that in patients with severe allergic reactions (anaphylaxis) to muscle relaxants a CD63 up-regulation (expressed in percentage of basophils showing CD63 expression) was observed in 57% of the cases, while all patients showed a down-regulation of the MFI-IgE. Similarly, 38% of patients with severe adverse reactions to non-steroidal anti-inflammatory drugs (NSAIDs) showed an increase of CD63 expression above the cutoff (positivity threshold) of 5% basophil activation, whereas a significant MFI-IgE down-regulation could be observed in 73% of the same patients. Specificities (control subjects below the cutoff point) were determined to be above 93% in both studies. A severe disadvantage of the method described in these latter two reports is the use of labelled anti-IgE antibodies primarily for positive selection of basophils which contrasts somewhat with its simultaneous use for following basophil activation as determined by IgE down-regulation.
Thus, there is a need for a more reliable method, which provides both high sensitivities and high specificities for drug induced basophil activation, taking into account that basophil activation is a complex process divided in early and late activation events and that basophil labelling with anti-IgE antibodies is currently used for basophil selection and not for determining so basophil activation.